Biotechnology, dealing with the preparation and use of biological systems, living or nonliving, in industrial applications is a rapidly expanding area of technology. One area of biotechnology involves the industrial or technological use of enzyme preparations which can be preferred, for example, in preparative or analytical chemistry, because of the great specificity possessed by enzymes. Such an increased use of enzymes requires that large quantities of relatively pure enzymes be available.
One such enzyme having industrial utility in alcohol oxidase which can be used in analytical or preparative chemistry involving one or more of the compounds of the following equation catalyzed by alcohol oxidase: ##STR1## wherein the abreviation A.O. indicates that the reaction occurs in the presence of alcohol oxidase, R is the alkyl group of 1, 2, 3, or more carbon atoms and R' is hydrogen or an alkyl group having 1, 2, or more carbon atoms so that R' has 1 fewer carbon atoms than R.
In order to follow the isolation of a single protein, some quantitative method of protein estimation is essential. In the case of purification of enzymes or protein hormones, estimation of specific activity of the enzyme is usually used to follow purification of the enzyme.
Enzymes can generally be purified, among others, by procedures such as the following: (1) differential solubility; (2) specific precipitation; (3) column chromatography; (4) preparative electrophoresis; and/or (5) preparative ultracentrifugation.
Methods of preparing alcohol oxidase such as, for example, the following are known in the prior art.
Janssen and Ruelius, 151 Biochem. Biophys. Acta 317 (1968) describe a method for production of crystalline alcohol oxidase using fractional precipitation with polyethylene glycol.
Tani et al., 36 Agr. Biol. Chem. 68 (1972) describe a method for preparation of crystalline alcohol oxidase using ammonium sulfate precipitation, column chromatography, and crystallization with solid ammonium sulfate.
Fujii and Tonomura, 36 Agr. Biol. Chem. 2297 (1972) describe a method for production of crystalline alcohol oxidase using ammonium sulfate precipitation and column chromatography.
Baratti et al., XX Biotech. and Bioeng. 333 (1978) describe a method for preparing immobilized alcohol oxidase by adsorption on DEAE cellulose, sometimes preceded by ammonium sulfate precipitation.
Purification of enzymes or of other natural products is an empirical procedure and the various methods are largely the result of trial of various procedures and materials known in the art to be effective as well as trial of new procedures and materials. At each stage of purification, the enzyme preparation must typically be monitored for (specific) activity or by physical examination. Testing enzyme activities for specific activity is generally time consuming and a fast accurate way of following active alcohol oxidase being purified without tests for specific activity is therefore highly desirable.
Further, even after alcohol oxidase preparation is sufficiently purified for the use to which it will be put, it is highly desirable to have an indicator of enzyme activity which does not require a measurement or observation of specific activity. Such an indicator could be included as a small part of a large quantity of alcohol oxidase and used to indicate the quality, i.e., activity or inactivity of the enzyme.
An object of the invention is a method and composition of matter for determining the presence of active alcohol oxidase without the necessity for observation of specific activity. Other objects of the invention are methods for purifying and isolating alcohol oxidase. Yet other objects and advantages of the instant invention will be apparent to one skilled in the art from the following description and the claims.